Herpesvirus-encoded protein kinases (HPKs) have attracted increasing attention lately due to the growing evidence of their involvement in different aspects of the viral life cycle, making them appealing targets for antiviral therapy. The broad objectives of this proposal are to study and exploit a virally encoded target for novel safe and effective antiviral agents against the human pathogen Epstein-Barr virus (EBV). The project takes a combined approach: the structure and properties of the EBV-encoded protein kinase (EBV-PK) will be studied by molecular biology and biochemistry techniques, and in search for the inhibitors both small-molecule and peptide-based strategies will be explored. The first specific aim is to study EBV-PK structure by mutagenesis, characterize its activity in regard to protein substrates and ganciclovir (GCV) and develop an assay for fast and effective screening of inhibitors. The activity of EBV-PK mutants will be studied both in vitro and in cell culture. The second specific aim is designed to study inhibition of the kinase by small-molecule and peptide-based inhibitors. Selected EBV-PK inhibitors will be further tested in cell culture for their ability to inhibit viral replication. Thus, this application aims to characterize a unique kinase, which is crucial for EBV replication, and to identify compounds that will efficiently inhibit this enzyme, potentially inhibiting the virus. Therefore it addresses a general biological question and a pharmaceutical problem. PUBLIC HEALTH RELEVANCE Given the growing number of conditions associated with EBV infection, many of which are notoriously difficult to treat, the development and testing of EBV-targeting therapy approaches is warranted in attempts to devise better treatments. The studies outlined in this proposal will advance understanding of EBV biology and evaluate a novel target for the development of the effective treatment of EBV-associated diseases.